Risuteganib Protects against Hydroquinone–induced Injury in Human RPE Cells

Abstract

PURPOSE. Cigarette smoking has been implicated in the pathogenesis of AMD. Integrin dysfunctions have been associated with AMD. Herein, we investigate the effect of risuteganib (RSG), an integrin regulator, on RPE cell injury induced by hydroquinone (HQ), an important oxidant in cigarette smoke. METHODS. Cultured human RPE cells were treated with HQ in the presence or absence of RSG. Cell death, mitochondrial respiration, reactive oxygen species production, and mitochondrial membrane potential were measured by flow cytometry, XFe24 analyzer, and fluorescence plate reader, respectively. Whole transcriptome analysis and gene expression were analyzed by Illumina RNA sequencing and quantitative PCR, respectively. F-actin aggregation was visualized with phalloidin. Levels of heme oxygenase-1, P38, and heat shock protein 27 proteins were measured by Western blot. RESULTS. HQ induced necrosis and apoptosis, decreased mitochondrial bioenergetics, increased reactive oxygen species levels, decreased mitochondrial membrane potential, increased F-actin aggregates, and induced phosphorylation of P38 and heat shock protein 27. HQ, but not RSG alone, induced substantial transcriptome changes that were regulated by RSG cotreatment. RSG cotreatment significantly protected against HQ-induced necrosis and apoptosis, prevented HQ-reduced mitochondrial bioenergetics, decreased HQ-induced reactive oxygen species production, improved HQ-disrupted mitochondrial membrane potential, reduced F-actin aggregates, decreased phosphorylation of P38 and heat shock protein 27, and further upregulated HQ-induced heme oxygenase-1 protein levels. CONCLUSIONS. RSG has no detectable adverse effects on healthy RPE cells, whereas RSG cotreatment protects against HQ-induced injury, mitochondrial dysfunction, and actin reorganization, suggesting a potential role for RSG therapy to treat retinal diseases such as AMD.

Targeting Lysosomes to Reverse Hydroquinone-Induced Autophagy Defects and Oxidative Damage in Human Retinal Pigment Epithelial Cells

“…Our results showed that cells incubated with HQ had marked disorganization of the mitochondrial network compared with control RPE cells (Figure 2A). Further, HQ exposure led to a significant reduction in the mitochondrial membrane potential of human RPE cells (Figure 2B), in agreement with recent studies [28]. While H 2 O 2 -treated cells also showed some changes in the mitochondrial network morphology (Figure 2A), the mitochondrial membrane potential remained unaffected after treatment with up to 500 µM H 2 O 2 (Figure 2B), confirming the relative resistance of RPE cells against H 2 O 2 -induced damage.…”

The Transcriptome Profile of Retinal Pigment Epithelium and Müller Cell Lines Protected by Risuteganib Against Hydrogen Peroxide Stress

“…Treatment with RSG alone was not evaluated because of the following: (1) it had a nonsignificant effect on cell viability and (2) previous study found it had a minimal effect on the transcriptome of human RPE cells. 24 RSG pretreatment followed by RSG and H 2 O 2 cotreatment was also not evaluated since both RSG pretreatment regimens showed comparable protection. All samples had excellent RNA quality and sequencing quality ( Supplementary Table S1 ).…”

“… 22 , 23 In a preclinical model of human RPE cells stressed with cigarette smoke toxin, RSG treatment reduced cell death and ROS level, upregulated cytoprotective genes, and improved mitochondrial bioenergetics and metabolic activity. 24 In RPE cells constructed to contain AMD donor mitochondria, RSG exposure reduced the expression of apoptosis and angiogenesis genes. 25 …”

Safety and Efficacy of Intravitreal Risuteganib for Non-Exudative AMD: A Multicenter, Phase 2a, Randomized, Clinical Trial

“…In human donor RPE cells, risuteganib was found to protect cells by reducing ROS level, upregulating cytoprotective heme oxygenase-1 protein level, and regulating genes in multiple disease-relevant pathways including inflammation, cell proliferation, cell adhesion and migration. 15 In RPE cells containing mitochondria derived from AMD patients, risuteganib exposure was associated with reduced expression of genes associated with apoptosis (BAX), angiogenesis (VEGFA) and integrin function (ITGB1). 16 Pharmacokinetics studies found risuteganib has a long half-life in the retina after intravitreal injection, whereas ex vivo studies suggest the peptide may be specifically localized to the RPE cells, where it may play a role in preserving and reversing the diseased cellular state.…”

“…17 This is further supported by studies from multiple labs that demonstrated improved mitochondrial bioenergetics and metabolic activity after risuteganib treatment, indicating potential reactivation of patient RPE with diminished mitochondrial function. 15,17,18 Studies of other antioxidants have also demonstrated reduction in retinopathy and improvement in visual parameters in animal models, [19][20][21] suggesting pro-mitochondrial therapeutics can lead to both morphological and functional improvements. Collectively, pre-clinical studies demonstrated that risuteganib has cytoprotective, antiinflammatory, and pro-mitochondria properties, which make it a potential candidate for use in the treatment of nonexudative AMD.…”